Human messenger RNA — the intermediate step between DNA and protein — is a bit like a choose-your-own-adventure book. Any book contains chapters arranged to tell a story. However, in a choose-your-own adventure, random chapters can be removed and the remaining sections stitched together in different combinations — and all of these new combinations tell a new story.
The process of taking out chapters, or sections of RNA, and putting what remains back together is called splicing, and it is performed by a molecular machine called the spliceosome. The lab of assistant professor Aaron Hoskins in the University of Wisconsin–Madison Department of Biochemistry is studying the inner workings of this complex machine to understand not only how it works but also how mutations of the spliceosome can lead to disease. Their recent findings published in Nucleic Acids Research could advance scientists’ understanding of how mutations in the spliceosome can lead to health problems like cancer.
In order to know which sections to cut out, the spliceosome recognizes unique parts of the RNA called introns. The leftover portions — called exons — are then joined together and continue down the path to becoming a protein. The spliceosome must do this with great precision to avoid ruining the information that the RNA contains.
“Splicing allows organisms like humans to encode greater genomic complexity without needing to dramatically increase the size of their genomes,” says Tucker Carrocci, a graduate student in the Hoskins Lab and lead author on the study. “Introns occur very frequently in the human genome with each gene having eight introns on average. These must be removed with high precision.”
This illustration gives an overview of spliceosome function. The multi-colored units make up the spliceosome, which cuts out the intron that sits between two exons. The exons are then joined together to continue on to becoming a protein. The mutations Carrocci investigated are in a protein in one of the colored sub-units.
Essentially, the spliceosome must recognize the boundaries of the intron in order to cut out the correct parts of the RNA without also cutting out portions that should remain. Imagine reading a choose-your-own-adventure book where the beginnings and endings of the chapters are just a page or two off. If the spliceosome is unable to do this due to a mutation, problems like cancer can arise.
Carrocci is part of the Integrated Program in Biochemistry (IPiB), the joint graduate program of the Department of Biochemistry and Department of Biomolecular Chemistry. His research focuses on the molecular function of the spliceosome component SF3b1.
“Mutations in this protein, SF3b1, have been linked with a number of human diseases, including some forms of leukemia,” he explains. “It’s not quite understood what the exact function of this protein is during splicing or how these mutations affect that function, and that is what we are trying to find out.”
The researchers explain that their findings could point to a need to re-evaluate some assumptions about cancer research in this area.
“There are many mutations that can affect SF3b1 and it was assumed that all of these mutations affect gene expression in generally the same way,” Hoskins says. “Our data suggest that may not be true, and we may need to take a more personalized medicine approach to treat these cancers.”
Using yeast, Carrocci and his fellow researchers introduced mutations in the spliceosome and then tested how well these mutated spliceosomes could excise introns from different RNAs. What the team ultimately learned is that the mutations cause the spliceosome to be unable to excise a subset of RNA introns properly. This disruption of the correct splicing can also disrupt production of the correct protein by the cell — potentially a cause for how these mutations can lead to cancer and other problems.
“These mutations affect the spliceosome in an unusual way,” Hoskins says. “It's really easy to make things that completely don't work. It is much harder to make things that work correctly some of the time and incorrectly at other times. And that's what these mutations do … the mutations in humans are changing a really basic step in gene expression.”
For this particular study, Carrocci and his colleagues — Doug Zoerner, a former undergrad now at the University of Kentucky medical school, and lab manager Josh Paulson — made hundreds of unique yeast strains and performed dozens of time intensive assays. The exact mechanisms that lead to cancer from these mutations are still unknown and deserve further study, the researchers say.
“I really enjoy learning how biological systems function and I think mutations are a really useful tool to learn more about biology,” Carrocci says. “The spliceosome has an enormous task in the cell, and yeast are a powerful way to learn what these mutations do.”